Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of phosphotyrosine, a central signal transduction control element. Small-molecule inhibitors that are specific for each cellular PTP would be valuable tools in dissecting phosphorylation networks. However, the common architecture of the conserved PTP protein fold impedes the discovery of selective PTP inhibitors. The broad objectives of the proposed research are to generate allele-specific inhibitors of PTPs, to validate their potency and selectivity in living cells, and to use the PTP inhibitors in mammalian cell-signaling experiments. Two distinct strategies- active-site engineering and WPD-loop targeting will be employed to generate PTPs that are uniquely sensitive to applied small-molecule inhibitors. In both approaches, functionally silent mutation(s) on a target PTP will sensitize the enzyme to a small molecule that does not inhibit wild-type PTPs. A significant advantage of these engineered-sensitivity approaches to PTP inhibition is that they can potentially yield general strategies for targeting multiple members of a large protein family- the amino-acid residues identified for sensitization are present across the protein family, eliminating the need to redesign a protein/inhibitor interface for each new PTP target. Once highly sensitizing mutations are discovered on model PTPs, primary-sequence alignments will allow for the identification of the corresponding positions in other PTPs, enabling the design, expression, and analysis of an array of sensitized PTPs for target-specific inhibition. Transfection of cells with genes encoding a sensitized PTP generates a biological system in which only one PTP can be blocked by the designed inhibitor, allowing for precise chemical control of a target PTP's activity in signaling studies. Improperly regulated PTP activity has been implicated as a causative agent in a range of human diseases, including leukemia, solid-tumor cancers, type I and type II diabetes, and a host of autoimmune disorders. A series of highly selective phosphatase inhibitors can be used to delineate the precise functions of each target PTP in signaling cascades and to validate PTPs as therapeutic targets. PUBLIC HEALTH RELEVANCE: Protein tyrosine phosphatases (PTPs) are enzymes that help to send cellular messages by enzymatically removing phosphate groups from other proteins. When cellular phosphate removal goes awry, so do the basic regulatory mechanisms of the cell, and improperly regulated PTP activity has been implicated as a causative agent in a range of human diseases, including cancer, diabetes, and autoimmune disorders. We are designing small-molecule inhibitors that can be used to study the functions of individual PTPs in cellular experiments;this work will thus provide valuable tools for dissecting cellular phosphorylation networks and for validating PTPs as potential therapeutic targets.